While proteins produced by a procaryote such as Escherichia coli and the like have no sugar chain, the proteins and lipids produced by an eucaryote such as yeast, fungi, plant cells, animal cells and the like have an attached sugar chain in many cases.
As a sugar chain in animal cells, N-linked sugar chain (also called as N-glycan) which binds to asparagine (Asn) residue in proteins, and O-linked sugar chain (also called as O-glycan) which binds to serine (Ser) or threonine (Thr) residue are known to be added to glycoproteins. There has been recently revealed that a certain lipids containing a sugar chain are bound covalently to a number of proteins and the proteins are attached to the cell membrane via those lipids. Those lipids containing a sugar chain are called as glycosyl phosphatidylinositol anchor.
The other example of a sugar chain in animal cells is glycosaminoglycan. A compound wherein a protein and a glycosaminoglycan are covalently bound is called as proteoglycan. Although glycosaminoglycan which is a component of sugar chain of proteoglycan has similar structure to that of O-glycan which is glycoprotein sugar chain, glycosaminoglycan-has-chemical properties different from those of O-glycan. Glycosaminoglycan has the characteristic structure composed of disaccharide unit repeats containing glucosamine or galactosamine and uronic acid (except that keratan sulfate has no uronic acid), wherein the sulfate groups are covalently bound thereto (except that hyaluronic acid has no sulfate groups).
Furthermore, as a sugar chain in animal cells, there is a sugar chain contained in glycolipid. As glycolipid in animal cells, there are known sphingoglycolipid in which sugar, long chain fatty acid and sphingosine which is long chain base are covalently bound, and glyceroglycolipid in which sugar chain is covalently bound to glycerol.
Recently, elucidation on the function of a sugar chain has been rapidly advanced together with advance in molecular biology and cell biology, and a variety of functions of a sugar chain have been revealed. Firstly, a sugar chain play an important role on clearance of glycoprotein in blood. Erythropoietin obtained by transferring a gene in Escherichia coli manifests in vitro its activity, but is known to be rapidly in vivo clearanced [Dordal et al.: Endocrinology, 116, 2293 (1985) and Browne et al.: Cold Spr. Harb. Symp. Quant. Biol., 51, 693, 1986]. Human granulocyte-macrophage colony stimulating factor (hGM-CSF) has naturally two N-linked sugar chains, but it is known that, as the-number of sugar chains is decreased, the clearance rate in rat plasma is raised proportionally thereto [Donahue et al.: Cold Spr. Harb. Symp. Quant. Biol., 51, 685 (1986)]. The clearance rate and clearanced places vary depending upon the structure of a sugar chain. It is known that, while hGM-CSF to which sialic acid is added is clearanced in kidney, hGM-CSF from which sialic acid is removed is raised in the clearance rate and is clearanced in liver. Additionally, the clearance rates in rat plasma and rat perfusion liquid were studied with respect .alpha.1-acid glycoproteins having different sugar chain which were biosynthesized by rat liver primary culture in the presence of various N-linked sugar chain biosynthesis inhibitors. In both cases, the clearance rates were slower in descending order of high mannose type, sugar chain deficient type, hybrid type and complex type (natural type). It is also known that the clearance in blood of tissue-type plasminogen activator (t-PA) used as a fibrinolytic agent is significantly influenced by the structure of a sugar chain.
It is also known that a sugar chain endows a protein with the protease resistance. For example, when formation of sugar chain of fibronectin is inhibited by zunicamycin, the resulting sugar chain deficient fibronectin is promoted in the intracellular degrading rate. It is also known that addition of a sugar chain increases the heat stability and anti-freezing properties. It is also known that a sugar chain makes a contribution to increase the solubility of protein.
A sugar chain also makes a contribution to holding the correct steric structure of proteins. It is known that, although the removal of two N-linked sugar chains naturally present in membrane-bound glycoprotein of vesicular stomatitis virus inhibits the transport of proteins to cell surface, new addition of a sugar chain to said proteins recovers the transport. In this case, it has been revealed that the removal of a sugar chain induces the association between protein molecules via disulfide bond and, as the result, the transport of proteins is inhibited. Since the correct steric structure is retained due to inhibition of this association by new addition of sugar chain, the transport of proteins becomes possible. It is shown that the position to which new sugar chain is added is considerably flexible. To the contrary, it has been revealed that the transport of proteins having natural sugar chains is completely inhibited, in some cases, depending upon the introduction site of the additional sugar chain.
There is known the case where a sugar chain masks antigen site on polypeptide. From the experiments using polyclonal antibody or monoclonal antibody reacting with a particular region on polypeptide in hGM-CSF, prolactin, interferon-.gamma., Rauscher leukemia virus gp70 and influenza hemagglutinin, it is considered that a sugar chain of the above proteins inhibits a reaction with antibody. There is also known the case where a sugar chain itself has direct relationship with manifestation of the activity of glycoprotein. For example, a sugar chain is considered to participate in manifestation of the activity of glycoprotein hormones such as luteinizing hormone, follicule stimulating hormone, chorionic gonadotropin and the like.
In addition, EP-A 0370205 discloses that granulocyte colony-stimulating factor (G-CSF), pro-urokinase (pro-UK) and the like can be improved in the properties by artificially and intentionally introducing a sugar chain into the proteins using recombinant DNA techniques.
Furthermore, as an important function of a sugar chain, there is participation in recognition phenomena between cells, between proteins or between cells and proteins. For example, it is known that the place where a sugar chain is clearanced in the living body is different depending upon the difference in the structure of a sugar chain. In addition, it has been found that a ligand of ELAM-1 which is inflammatory response-specifically expressed on blood vessel endothelial cell and promotes adhesion to neutrophil is a sugar chain called as Sialyl-Le.sup.X [NeuAc .alpha.2.fwdarw.3Gal .beta.1.fwdarw.4 (Fuc .alpha.1.fwdarw.3)GlcNAc:NeuAc, sialic acid; Gal, galactose; Fuc, fucose; GlcNAc, N-acetylglucosamine]. As the result, there has risen the possibility that a sugar chain itself or-modification thereof is used in pharmaceuticals and the like [Phillips et al.: Science 250, 1130 (1990), Goelz et al.: Trends in Glycoscience and Glycotechnology 4, 14 (1992)]. Further, it is suggested that L-selectin which is expressed in a part of T lymphocytes and neutrophil and GMP-140 (also called as P-selectin) which is expressed in the membrane surface of platelet and blood vessel endothelial cell by inflammatory stimulation participate in inflammatory response as same as ELAM-1, and ligands thereof are also sugar chains similar to Sialyl-Le.sup.X sugar chain which is a ligand of ELAM-1 [Rosen et al.: Trends in Glycoscience and Glycotechnology 4, 1 (1992), Larsen et al.: Trends in Glycoscience and Glycotechnology) 4, 25 (1992), Aruffo et al.: Trends in Glycoscience and Glycotechnology 4, 146 (1992)].
Also in metastasis of cancer as in the inflammatory response, it is suggested that ELAM-1 and GMP-140 promote metastasis of cancer by causing adhesion of cancer cells to inner wall of blood vessel or aggregation between cancer cells and platelets [Goelz et al.: Trends in Glycoscience and Glycotechnology) 4, 14 (1992), Larsen et al.: Trends in Glycoscience and Glycotechnology) 4, 25 (1992)]. These suggestions are consistent with the findings that expression of Sialyl-Le.sup.X sugar chain is high in cancer cells having high metastasis ability [Irimura et al.: Experimental Medicine 6, 33 (1988)].
From these findings, it is expected that Sialyl-Le.sup.X sugar chain or derivatives thereof manifest the excellent anti-inflammatory effects and anti-metastatic effects by binding to ELAM-1, L-selectin or GMP-140.
Additionally, in view of the mechanism of the above-described inflammatory response and metastasis of cancer, inflammatory response could be inhibited and metastasis of cancer could be prevented by inhibiting the expression of glycosyltransferase which controls synthesis of ligand sugar chain recognized by ELAM-1, L-selectin or GMP-140. Antisense RNA/antisense DNA techniques [Tokuhisa: Bioscience and Industry 50, 322 (1992), Murakami: Chemistry 46, 681 (1991)] or Triple helix techniques [Chubb and Hogan: Trends in Biotechnology 10, 132 (1992)] are useful for inhibiting the expression of some particular genes. Since information on those gene or nucleotide sequence of those gene is necessary in order to inhibit the expression of desired glycosyltransferase using this antisense RNA/DNA techniques, the cloning of a gene of desired glycosyltransferase and analysis of the information on the nucleotide sequence are important.
Further, diagnosis of malignancy in inflammatory diseases or cancer can be also performed by investigating the expression of the particular glycosyltransferase in inflammatory lymphocyte and cancer cells. For investigating the expression of desired glycosyltransferase, Northern hybridization method which uses as a probe the relevant gene labelled with radioactivity and the like [Sambrook, Fritsch, Maniatis, Molecular Cloning, A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989] and Polymerase Chain Reaction method (abbreviated as PCR method hereinafter) [Innis et al.: PCR Protocols, Academic Press, 1990] are useful. For applying these methods, the information on desired glycosyltransferase gene or nucleotide sequence thereof is necessary. Also from this respect, the cloning of desired glycosyltransferase gene and analysis of information of that nucleotide sequence are important.
As described above, alteration in the structure of glycoprotein and mass production of the particular sugar chain or modification thereof are industrially extremely important themes.
The means by which the structure of a sugar chain is altered have recently advanced remarkably. In particular, the structure of a sugar chain can be altered by a high specific enzyme (exoglycosidase) which successively dissociates a sugar chain or glycopeptidase and endo-type glycosidase which cleavages a bonding between peptide chain and sugar chain without changing both peptide chain and sugar chain. As the result, the biological role of a sugar chain can be studied in detail. Further, endoglycoceramidase which cleavages between a sugar chain of glycolipid and ceramide has been recently found [Ito and Yamagata: J. Biol. Chem. 261, 14278 (1986)]. This finding has not only facilitated the preparation of a sugar chain of glycolipid but also advanced the study on the function of cell surface glycolipid. In addition, new addition of sugar chain has been possible by using glycosyltransferase. For example, sialic acid can be newly added to the end of sugar chain by using sialyltransferase [Sabesan and Paulson: J. Am. Chem. Soc. 108, 2068 (1986)]. A sugar chain to be added can be varied by using other various glycosyltransferases or glycosidase inhibitors [Alan et al.: Annu. Rev. Biochem. 56, 497 (1097)]. However, mass production of glycosyltransferase used in synthesis of a sugar chain is extremely difficult. For that reason, it is desired that glycosyltransferase is produced in a large amount by cloning glycosyltransferase using recombinant DNA techniques and effectively expressing glycosyltransferase in a host cell.
As a method for cloning glycosyltransferase, there are known a method by purifying a protein, produing an antibody reacting with it and performing immunoscreening using the antibody [Weinstein et al.: J. Biol. Chem. 262, 17735 (1987)], and a method by purifying a protein, determining amino acid sequence thereof, producing synthetic DNA which corresponds thereto and performing hybridization using the DNA as a probe [Narimatsu et al.: Proc. Natl. Acad. Sci., USA, 83, 4720 (1986)]. A method is also known where hybridization is performed using cloned glycosyltransferase gene as a probe and thereby glycosyltransferase gene having homology with the glycosyltransferase is cloned [John. B. Lowe et al.: J. Biol. Chem. 266, 17467 (1991)]. In addition, there is known a cloning method by direct expression cloning using panning method as screening method, in which antibody or lectin reacting with a sugar chain is employed [John. B. Lowe et al.: Proc. Natl. Acad. Sci.,USA, 86, 8227 (1989), John. B. Lowe et al.: Genes Develop., 4, 1288 (1990)].
There is no case where glycosyltransferase can be cloned using lectin-resistance as an index. From the studies on various lectin-resistant mutants of CHO cell, it has been revealed that there are cases where new glycosyltransferase is expressed, where the activity of a certain glycosyltransferase disappears, and where synthesis of sugar nucleotide or its transfer to Golgi body is inhibited [Pamela Stanley et al.: Methods in Enzymology, 96, 157]. Therefore, it is considered that cloning of glycosyltransferase can be performed using lectin-resistance as index by introducing a gene derived from a cell expressing glycosyltransferase to be cloned into CHO cell or lectin-resistant mutants of CHO cell [Ravindra Kumar et al.: Mol. Cell. Biol., 9, 5713 (1989)]. James Ripka et al. have tried to clone N-acetylglucosaminyltransferase I by introducing human genomic DNA derived from A431 cell into lectin-resistant mutants of CHO cell (Lecl) using resistance to lectin concanavalin A as an index. However, they could not clone glycosyltransferase by the screening method using lectin-resistance as an index [James Ripka et al.: Biochem. Biophys. Res. Commun., 159, 554 (1989)]. Heffernan et al. have cloned mouse sialic acid hydroxylase using resistance to lectin WGA (wheat germ agglutinin) as an index by introducing cDNA library into CHO cell [Michael Heffernan et al.: Nucleic Acids Res., 19, 85 (1991)] which was made to produce large T antigen of polyoma [Michael Heffernan et al.: Glycoconjugate J., 8, 154 (1991)]. However, there is no report in which glycosyltransferase could be cloned in a screening system using the lectin-resistance as an index. In addition, with respect to hosts, Stanley, Ripka, Heffernan et al. all used CHO cell or lectin-resistant mutants of CHO cell as a host.
With respect to sialyltransferase, a cDNA encoding an enzyme having .beta.galactoside .alpha.2.fwdarw.6 sialyltransferase activity has been isolated and nucleotide sequence thereof has been revealed [Weinstein et al.: J. Biol. Chem., 262, 17735 (1987)]. With respect to an enzyme having .beta.galactoside .alpha.2.fwdarw.3 sialyltransferase activity, Gillespie et al. have reported cloning of a gene encoding an enzyme which adds sialic acid to galactose in O-linked sugar chain of glycoprotein (sugar chain which is added to serine or threonine residue), but base sequence thereof has not been revealed [Gillespie et al.: Glycoconjugate J., 7, 469 (1990)]. In addition, Weinstein et al. have been reported a method for purifying an enzyme having .beta.galactoside .alpha.2.fwdarw.3 sialyltransferase activity from rat liver [Weistein et al.: J. Biol. Chem., 257, 13835 (1982)]. However, desired enzyme can be obtained in an extremely small amount. Hitherto, there have been no reports in which sialic acid is added in .alpha.2.fwdarw.3 linkage to desired position on sugar chain of glycoprotein, glycolipid, oligosaccharide and the like using recombinant DNA techniques.